Periodic Reporting for period 1 - ReCNNSTRCT (Reconstructing enzymes for novel nitrogen-nitrogen bond forming chemistry)
Okres sprawozdawczy: 2023-01-01 do 2025-06-30
Biocatalysis uses enzymes to replace traditional catalysts. This technology has already established sustainable enzymatic approaches in several industrial sectors, resulting in economic and environmental benefits. Despite the growing impact of biocatalysis in industrial chemistry, the full potential of this technology has yet to be realized, particularly for reactions for which effective biocatalysts are not yet available. Nitrogen-nitrogen bond forming enzymes (NNzymes) are one such group. They catalyze unique chemical reactions, but are vastly underexplored as sustainable biocatalysts in the chemical industry. Although the native reactions of NNzymes have recently been investigated, the lack of knowledge on how these enzymes perform the challenging nitrogen-nitrogen (N-N) bond formation hinders a systematic exploration of their reactivity towards non-natural substrates and transformations. This knowledge gap has prevented the development of versatile N-N enzyme platforms, especially for reactions not observed in nature. The ReCNNSTRCT project, funded by the European Research Council, aims to overcome these challenges by exploiting the synthetic potential of NNzymes for the sustainable selective production of high-value compounds such as pharmaceuticals, dyes or agrochemicals through N-N bond formation.
In the first 2 years of this project, we have made great progress towards our ambitious research vision of establishing N-N bond forming enzymes as versatile biocatalysts in organic synthesis (ACS Catal. 2025, 15, 310-342, 10.1021/acscatal.4c05268). We have generated libraries of NNzymes from three different families by genome mining and identified new interesting candidates for further biocatalytic applications. To unravel the potential of candidate enzymes, we extensively screened them by simple plate assays or LC/GC-MS analysis for substrate scope and identification of potential products/reactions. To demonstrate substrate promiscuity in the piperazate synthase (PZS) family, we analyzed various N-hydroxylated diamines as substrates other than the natural substrate. The N-hydroxylated diamines were obtained in situ using a panel of N-hydroxylating monooxygenases (NMOs), allowing subsequent cyclization by PZS to yield various N-N bond-containing heterocycles. The screened panel yielded 17 hydroxylated diamines and new promiscuous NMOs, thereby expanding the substrate set of poorly accessible hydroxylated products. The investigated PZSs led to a series of 5- and 6-membered N-N bond-containing heterocycles, and the most promiscuous catalysts were used to scale up and optimize the synthesis, yielding the desired N-N bond-containing heterocycles with up to 45% isolated yield (DOI: 10.26434/chemrxiv-2025-f1l90). Overall, our data provide essential insights into the substrate promiscuity and activity of NMOs and PZSs, further enhancing the potential of these biocatalysts for an expanded range of N-N coupling reactions.
Screening of the remaining NNzymes from our libraries also revealed an unexpectedly broad substrate range, leading to additional N-N bonded products as pharmaceutical building blocks (unpublished data). In parallel, we were able to perform structural and mechanistic studies based on either structural models generated with AlphaFold or X-ray crystallography, and developed biocatalytic cascades involving NNzymes to demonstrate their synthetic potential for pharmaceutical synthesis (unpublished data).
Overall, in the first years of this ERC project, we have made significant progress in establishing NNzymes as versatile biocatalysts for applications in organic synthesis. The NNzyme candidates identified so far in our toolbox provide access to cyclic hydrazines, aromatic diazo compounds and aromatic hydrazones, thus offering a novel biocatalytic methodology to access these precursors. In particular, PZS provide a particularly attractive route to the synthesis of hydrazines as the enzymes do not require any cofactor and produce only water is a by-product of the reaction.
Screening of the remaining NNzymes from our libraries also revealed an unexpectedly broad substrate range, leading to additional N-N bonded products as pharmaceutical building blocks (unpublished data). In parallel, we were able to perform structural and mechanistic studies based on either structural models generated with AlphaFold or X-ray crystallography, and developed biocatalytic cascades involving NNzymes to demonstrate their synthetic potential for pharmaceutical synthesis (unpublished data).
Overall, in the first years of this ERC project, we have made significant progress in establishing NNzymes as versatile biocatalysts for applications in organic synthesis. The NNzyme candidates identified so far in our toolbox provide access to cyclic hydrazines, aromatic diazo compounds and aromatic hydrazones, thus offering a novel biocatalytic methodology to access these precursors. In particular, PZS provide a particularly attractive route to the synthesis of hydrazines as the enzymes do not require any cofactor and produce only water is a by-product of the reaction.
In the first two years of this project, we have shown that NNzymes offer unprecedented potential for applications in organic synthesis to access a variety of N-N bonded compounds of particular interest as pharmaceutical building blocks. These studies have opened up exciting new avenues in biocatalysis that will be fully explored in the remainder of the research project. Key areas of ongoing and future investigation include the continued screening of novel NNzyme functions against non-natural substrates and reactivities; a detailed understanding of structure-function relationships and mechanistic features elucidating how the challenging N-N bond formation is achieved by NNzymes; the development of NNzymes into broadly applicable biocatalysts with high activity and stability; the use of high-throughput screening techniques to rapidly engineer NNzymes with an extended substrate range; and the development of biocatalytic cascades involving NNzymes to access multifunctionalized products containing the N-N bond motif. The realization of these goals will allow the field of biocatalysis to move beyond the state of the art and implement versatile NNzyme platforms, especially for reactions not observed in nature.